Measurement of hydrogen ion concentration using ratiometry and utilizing oblique illumination for structural orientation measurement
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Fluorescence is the phenomenon in which absorption of light of a given wavelength by a fluorescent molecule is followed by the emission of light at longer wavelengths. Ratiometry refers to a technique for measuring ion concentration in which two images are obtained at different wavelengths, and a ratio image is obtained by dividing corresponding pixels in each image. These fluorescence intensity values were entered in the look up table (LUT) with the corresponding wavelength and ion concentration from the chart. The hydrogen ion concentration of the ratio image is obtained with the help of the graph drawn between fluorescence excitation ratio and calcium ion concentration from the LUT. Birefringence spectroscopy is the optical technique of measuring orientation in an optically anisotropic sample by measuring the retardation of polarized light passing through the sample. Oblique illumination is used where illumination in a microscope is directed so that it originates from a single azimuth and strikes the specimen from an oblique angle, details in the specimen may be revealed with much greater contrast and visual clarity. This method of Oblique Illumination is used to calculate Birefringence.